3-oxo-C12:2-HSL, quorum sensing molecule from human intestinal microbiota, inhibits pro-inflammatory pathways in immune cells via bitter taste receptors.

In the gut ecosystem, microorganisms regulate group behaviour and interplay with the host via a molecular system called quorum sensing (QS). The QS molecule 3-oxo-C12:2-HSL, first identified in human gut microbiota, exerts anti-inflammatory effects and could play a role in inflammatory bowel diseases where dysbiosis has been described. Our aim was to identify which signalling pathways are involved in this effect. We observed that 3-oxo-C12:2-HSL decreases expression of pro-inflammatory cytokines such as Interleukine-1ß (- 35%) and Tumor Necrosis Factor-α (TNFα) (- 40%) by stimulated immune RAW264.7 cells and decreased TNF secretion by stimulated PBMC in a dose-dependent manner, between 25 to 100 µM. Transcriptomic analysis of RAW264.7 cells exposed to 3-oxo-C12:2-HSL, in a pro-inflammatory context, highlighted JAK-STAT, NF-κB and TFN signalling pathways and we confirmed that 3-oxo-C12:2-HSL inhibited JAK1 and STAT1 phosphorylation. We also showed through a screening assay that 3-oxo-C12:2-HSL interacted with several human bitter taste receptors. Its anti-inflammatory effect involved TAS2R38 as shown by pharmacologic inhibition and led to an increase in intracellular calcium levels. We thus unravelled the involvement of several cellular pathways in the anti-inflammatory effects exerted by the QS molecule 3-oxo-C12:2-HSL.

Reconstruction of functional human epidermis equivalent containing 5%IPS-derived keratinocytes treated with mitochondrial stimulating plant extracts.

Reconstructed human epidermis equivalents (RHE) have been developed as a clinical skin substitute and as the replacement for animal testing in both research and industry. KiPS, or keratinocytes derived from induced pluripotent stem cells (iPSCs) are frequently used to generate RHE. In this study, we focus on the mitochondrial performance of the KiPS derived from iPSCs obtained from two donors. We found that the KiPS derived from the older donor have more defective mitochondria. Treatment of these KiPS with a plant extract enriched in compounds known to protect mitochondria improved mitochondrial respiration and rendered them fully competent to derive high-quality RHE. Overall, our results suggest that improving mitochondrial function in KiPS is one of the key aspects to obtain a functional RHE and that our plant extracts can improve in this process.

Enhancing cell longevity for cosmetic application: a complementary approach.

BACKGROUND AND OBJECTIVES: Cell longevity is linked to sirtuins (silent information regulators), which belong to a family of enzymes implicated in gene silencing, apoptosis, fatty acid metabolism, and regulation of cellular life spans of organisms. Sirtuins are associated with genes that coordinate and optimize the functions of cells as cells struggle to survive in a stressful environment, as it is the case for skin cells. This study focuses on 1) yeast Kluyveromyces biopetides in stimulating the expression of sirtuin in human cutaneous cells and 2) the benefit for the skin of an active skin care product containing yeast Kluyveromyces biopetides. METHODS: Silent mating type information regulation 2 homolog 1 (SIRT1) was investigated by immunostaining, Westem blotting, and cytometry on normal human skin cells in culture and on healthy skin samples ex vivo. SIRT7 are mammalian versions of the yeast SIR2 gene. Cellular integrity and aging was followed by comet assays measuring DNA fragmentation and beta galactosidase activity (a marker of senescence). The test product was yeast Kluyveromyces biopeptides. Thirty-three female subjects aged 37 to 64 years (mean 51.6 years) enrolled in the study. Subjects applied a formulation enriched in 1% of the yeast biopeptides SIRT1 activator once daily to the face and neck for 4 weeks. Dermatologists used a graded scale (1-9) to score fine lines and wrinkles, hydration, pigment color intensity, complexion radiance, skin density, firmness, complexion homogeneity, and texture of the skin before and after the first application and again after 4 weeks of use. A Pixel Skin method, based on an analysis of the gray-level variance and surface of imperfections (age-related parameters) from numerical pictures of the faces, was used to objectively measure the skin care efficacy. RESULTS: The yeast Kluyveromyces biopeptides 1) significantly increased SIRT1 expression in normal human dermal skin fibroblasts in vitro (+172%) and in epidermal cells of healthy human skin ex vivo and 2) decreased cell senescence and DNA fragmentation induced by ultraviolet-B (UVB) stress. At the end of the study, facial improvements could be seen on fine lines and wrinkles, hydration, pigmented spot color intensity, complexion radiance, firmness, complexion homogeneity, and texture. Improvement in hydration was significant immediately after the first application. Skin-pixel measurement and analysis show a significant reduction of the gray variance linked to pixel heterogeneity (-4.2%) and a significant reduction of the surface of skin imperfections (-30.4%). All the indicators from clinical evaluation to the objective measurements of the skin show a significant improvement of the aged skin. CONCLUSION: These results demonstrate the efficacy of the yeast Kluyveromyces biopeptides in activating SIRT 1 of human skin cells, improving their DNA resistance and senescence, and of a formulation enriched in this ingredient in treating multiple skin aging signs.

Impairment of methionine sulfoxide reductase during UV irradiation and photoaging.

During chronic UV irradiation, which is part of the skin aging process, proteins are damaged by reactive oxygen species resulting in the accumulation of oxidatively modified protein. UV irradiation generates irreversible oxidation of the side chains of certain amino acids resulting in the formation of carbonyl groups on proteins. Nevertheless, certain amino acid oxidation products such as methionine sulfoxide can be reversed back to their reduced form within proteins by specific repair enzymes, the methionine sulfoxide reductases A and B. Using quantitative confocal microscopy, the amount of methionine sulfoxide reductase A was found significantly lower in sun-exposed skin as compared to sun-protected skin. Due to the importance of the methionine sulfoxide reductase system in the maintenance of protein structure and function during aging and conditions of oxidative stress, the fate of this system was investigated after UVA irradiation of human normal keratinocytes. When keratinocytes are exposed to 15 J/cm(2) UVA, methionine sulfoxide reductase activity and content are decreased, indicating that the methionine sulfoxide reductase system is a sensitive target for UV-induced inactivation.

Proteasome and photoaging: the effects of UV irradiation.

Cellular aging is characterized by the accumulation of oxidatively modified proteins that result, at least in part, from impaired degradation of abnormal proteins. The proteasome is the major intracellular proteolytic system implicated in the removal of abnormal and oxidized proteins. In human epidermal cells, previous studies have evidenced that proteasome function is decreased during aging as well as upon UV irradiation, which is the main component of photoaging. The age-related decline of proteasome activity has been reported to be due to either or both decreased proteasome subunits expression and content, inactivation upon alteration of proteasome subunits, and accumulation of endogenous inhibitors, such as highly oxidized and cross-linked proteins. To gain further insight in the mechanisms that might be implicated in the decreased activity of the proteasome upon photoaging, purified 20S human proteasome has been exposed to UVA- and UVB-irradiation. The effect of such an irradiation on proteasome peptidase activities has been monitored and shown to promote a stimulation or an inhibition of the peptidase activities depending on whether the proteasome is under its latent or a nonphysiological active form. Analysis of the patterns of proteasome subunits by 2D gel electrophoresis has revealed modification for several subunits for UV-irradiated proteasome only in its irreversibly activated form, compared with nonirradiated and irradiated latent forms, indicating that the 20S proteasome is rather resistant to UV irradiation.

Algae extract-mediated stimulation and protection of proteasome activity within human keratinocytes exposed to UVA and UVB irradiation.

Sun exposure is the major environmental influence for epidermal cells; the harmful effect of UV radiation on skin is related to the generation of reactive oxygen species that alter cellular components including proteins. It is now well established that the proteasome is responsible for the degradation of most of oxidized proteins and that impairment of proteasome function is a hallmark of cellular aging. In a previous study, we investigated the effects of UV irradiation on proteasomes in human keratinocyte cultures and showed that all three peptidase activities were decreased 24 h after irradiation of the cells. Increased levels of oxidatively modified proteins were observed in irradiated cells and were found to act as endogenous inhibitors of the proteasome. We report here on the stimulating and protective effects of an algae extract, prepared from Phaeodactylum tricornutum, on proteasome peptidase activities of human keratinocytes exposed to UVA and UVB irradiation. In addition, preserving proteasome function resulted in lowering the extent of the irradiation-induced protein oxidative damage, opening up new strategies for protection of epidermal cells against the detrimental effects of UV irradiation.

Overexpression of MsrA protects WI-38 SV40 human fibroblasts against H2O2-mediated oxidative stress.

Proteins are modified by reactive oxygen species, and oxidation of specific amino acid residues can impair their biological functions, leading to an alteration in cellular homeostasis. Oxidized proteins can be eliminated through either degradation or repair. Repair is limited to the reversion of a few modifications such as the reduction of methionine oxidation by the methionine sulfoxide reductase (Msr) system. However, accumulation of oxidized proteins occurs during aging, replicative senescence, or neurological disorders or after an oxidative stress, while Msr activity is impaired. In order to more precisely analyze the relationship between oxidative stress, protein oxidative damage, and MsrA, we stably overexpressed MsrA full-length cDNA in SV40 T antigen-immortalized WI-38 human fibroblasts. We report here that MsrA-overexpressing cells are more resistant than control cells to hydrogen peroxide-induced oxidative stress, but not to ultraviolet A irradiation. This MsrA-mediated resistance is accompanied by a decrease in intracellular reactive oxygen species and is partially abolished when cells are cultivated at suboptimal concentration of methionine. These results indicate that MsrA may play an important role in cellular defenses against oxidative stress, by catalytic removal of oxidant through the reduction of methionine sulfoxide, and in protection against death by limiting, at least in part, the accumulation of oxidative damage to proteins.

Algae extract protection effect on oxidized protein level in human stratum corneum.

Modification of proteins by reactive oxygen species is implicated in different disorders. The proteasome is a multicatalytic proteinase in charge of intracellular protein turnover and of oxidized proteins degradation. Consequently, proteasome function is very important in controlling the level of altered proteins in eukaryotic cells. Evidence for a decline in proteasome activity during skin photo-aging has been provided in Bulteau et al. in 2002. The ability of a lipid algae extract (Phaeodactylum tricornutum) to stimulate 20S proteasome peptidase activities was described by Nizard et al. in 2001. Furthermore, keratinocytes treated with Phaeodactylum tricornutum extract and then UVA and UVB irradiated, exhibited a sustained level of proteasome activity comparable to the one of nonirradiated cells. The level of modified proteins can be quantified by measurement of protein carbonyl content (Oxyblot technique), which has been shown to increase with aging and other disorders. In this paper, it is described that, in the presence of this lipid algae extract, the level of oxidized proteins is reduced, as assessed by the Oxyblot technique. These results are obtained both with culture of human keratinocytes and stratum corneum skin cells (obtained by stripping) from human volunteers. Altogether, these results argue for the presence of compounds in this algae extract that have a stimulating and/or protective effect on proteasome activity, resulting in a decreased level of protein oxidation.

Heat shock protein 47 expression in aged normal human fibroblasts: modulation by Salix alba extract.

Heat shock protein (HSP) 47 is a specific chaperone of procollagen. This heat shock protein is responsible for the correct three-dimensional organization of procollagen and its control-quality prior secretion. The aim of the study is to evaluate the level of HSP 47 in aged, photoaged, and senescent fibroblasts and its modulation by a plant extract (Salix alba). The level of HSP 47 and/or procollagen expression in fibroblasts was measured by real-time RT-PCR (mRNA transcripts) and by flow cytometry (immunochemistry technique for measurement of arbitrary fluorescence intensity). Immunochemistry techniques and confocal microscopy were used to visualize the cellular localization of HSP 47 and procollagen. These parameters were compared with different age donors, nonsenescent, and senescent fibroblasts. Fibroblasts were irradiated by a noncytotoxic dose of UVA (6 J/cm(2)), and HSP 47 level was evaluated. S. alba extract was tested for its capacity to modulate HSP 47 expression. Colocalization of HSP 47 and procollagen was shown by confocal microscopy, indicating that HSP 47 could play a role of procollagen molecular chaperone in the cellular model. It was also shown that the HSP 47 level is decreased in old-donor cells, senescent, and irradiated cells. This decrease can be modulated by a S. alba extract (polyphenols rich) in a dose-dependent manner. The evaluation of HSP 47 expression in the experimental conditions can lead to a new approach of aging and photoaging, pointing out the implication of this chaperone in these pathophysiologic phenomena. Modulation of HSP 47 expression by this family of molecules could be of cosmetic and/or dermatologic interest.

Ultraviolet A radiation transiently disrupts gap junctional communication in human keratinocytes.

Ultraviolet A (UVA) (320-400 nm) radiation is known to cause cutaneous aging and skin cancer. We studied the effect of UVA (365 nm) radiation on the human epidermis by focusing on keratinocyte gap junction-mediated intercellular communication (GJIC). We observed a dose-dependent 10-fold decrease in GJIC induced by UVA in normal human keratinocytes. This decrease in GJIC was associated with time-dependent internalization of connexin43 (Cx43). UVA radiation also damaged the actin cytoskeleton, as shown by microfilament disappearance. Importantly, the decrease in GJIC was transient when keratinocytes were irradiated with 10 J/cm(2) UVA, with a return to baseline values after 8 h. Concomitantly, Cx43 was relocalized and the actin cytoskeleton was restored. UVA irradiation and 12-O-tetradecanoylphorbol 13-acetate (TPA) treatment activated protein kinase C and reduced GJIC. However, Cx43 localization and phosphorylation were differently regulated by the two treatments. This suggests that at least two different pathways may mediate the observed fall in GJIC. These findings identify keratinocyte GJIC as a new UVA target that might sensitize human skin to photoaging and cancer formation.

Impairment of proteasome function upon UVA- and UVB-irradiation of human keratinocytes.

The major environmental influence for epidermal cells is sun exposure and the harmful effect of UV radiation on skin is related to the generation of reactive oxygen species that are altering cellular components including proteins. It is now well established that the proteasome is responsible for the degradation of oxidized proteins. Therefore, the effects of UV-irradiation on proteasome have been investigated in human keratinocyte cultures. Human keratinocytes were irradiated with 10 J/cm(2) of UVA and 0.05 J/cm(2) of UVB and proteasome peptidase activities were measured in cell lysates using fluorogenic peptides. All three peptidase activities were decreased as early as 1 h and up to 24 h after irradiation of the cells. Increased levels of oxidized and ubiquitinated proteins as well as proteins modified by the lipid peroxidation product 4-hydroxy-2-nonenal were also observed in irradiated cells. However, immunopurified 20S proteasome exhibited no difference in both peptidase specific activities and 2D gel pattern of subunits in irradiated cells, ruling out the possibility that the 20S proteasome could be a target for the UV-induced damage. Finally, extracts from irradiated keratinocytes were able to inhibit degradation by the proteasome, demonstrating the presence of endogeneous inhibitors, including 4-hydroxy-2-nonenal modified proteins, generated upon UV-irradiation.